Formalin-fixed paraffin embedded (FFPE) samples are derived from tissues (usually suspected tumor samples obtained by biopsy or dissection) that are fixed with formalin to preserve the cytoskeletal and protein structure and then embedded in a type of paraffin wax. FFPE is well-established and is widely used for histological analysis of clinical and research tissue samples utilized by pathologists for diagnostic purposes and by researchers. FFPE has been the preferred method for preserving tissue samples because it allows for extended room temperature storage of tissues while maintaining histopathology integrity. DNA, RNA, and proteins have been successfully extracted from bulk FFPE samples.
Formalin serves to cross-link proteins thus preserving the structural integrity of the cells and permitting the cells to be examined for histological, pathological, or cytological studies. FFPE samples are commonly sliced on a microtome, which is an instrument used to prepare very thin slices of a sample.
A conventional method for removing a cell or a small group of cells from a FFPE sample is laser capture microdissection (LCM). LCM combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that need to be separated from others. Two conventional LCM systems are schematically illustrated in FIGS. 1A and 1B. FIG. 1A schematically illustrates the PixCell II infrared LCM system commercialized by Arcturus Engineering (Mountain View, Calif., US), which utilizes a thin transparent thermoplastic film 10 (supported by a collector 16) that is placed over a tissue section supported by a glass slide 12. Cells of interest 14 are selectively adhered to the film 10 with a fixed-position, short duration, focused IR laser pulse 24. The adherence of the cells of interest 14 to the film 10 exceeds the adhesion to the glass slide 12, thereby allowing selective removal of cells of interest 14. Such cells of interest 14 are detached by lifting the film 10, and are transferred to an Eppendorf microcentrifuge tube (not shown) containing a buffer solution for isolation of DNA or RNA. FIG. 1B schematically illustrates the Palm Zeiss ultraviolet LCM system commercialized by P.A.L.M. Microlaser Technologies AG (Bernried, Germany). A highly focused UV laser beam 22 is used to cut out cells or regions of interest 14 from a tissue sample supported by a glass slide 12. By increasing the power of the laser, desired cells of interest 14 are ejected against gravity into a collection device (a “cap”) 18 containing a lysis buffer 20.
LCM can cut single cells from planar (two-dimensional) FFPE tissues, but cannot remove cells individually that are stacked on top of each other. In other words, LCM lacks resolution in the z-direction. LCM methods also do not lend themselves to high throughput since each cut cell must be placed into an Eppendorf microcentrifuge tube and subsequently handled.
The art continues to seek improved methods and apparatuses for extracting and collecting single cells or small groups of cells from FFPE tissue samples to overcome limitations associated with conventional methods and apparatuses. Aspects of this disclosure address shortcomings associated with conventional systems and methods.